Many enzyme- catalyzed reactions involve abstraction of a weakly acidic proton from the carbon adjacent to a carbonyl or carboxylic acid group (alpha-proton of a carbon acid) by a weakly basic active site general basic catalyst. An electrophilic (general acidic) catalyst positioned adjacent to the carbonyl group of the substrate so that an enolic intermediate can be stabilized by a short, strong hydrogen bond to the acid catalyst allows a quantitative understanding of the rates and mechanisms of these reactions. Dr. Gerlt will investigate the importance of electrophilic catalysis in three enzyme-catalyzed reactions: 1) the cycloisomerization of cis,cis-muconate catalyzed by muconate lactonizing enzyme I (MLE I); 2) the beta-elimination of water from acid sugars catalyzed by galactonate dehydratase (GalDH) and glucarate dehydratase (GlucDH); and 3) the FMN-dependent oxidation of S-mandelate catalyzed by S-mandelate dehydrogenase (MDH). MLE I, GalDH, and GlucDH are homologous to the structurally and mechanistically characterized mandelate racemase (MR). The proposed studies will increase our understanding of the rates and mechanisms of proton abstraction from carbon acids, in general, and beta-elimination reactions, in particular. Although both MR and MDH catalyze the abstraction of the alpha proton of S-mandelate to generate enolic intermediates, the active site architectures of MR and MDH are significantly different, e. g., the reaction catalyzed by MR but not MDH is absolutely dependent upon Mg2+. The investigator has been able to detect the enolic intermediate in the reaction catalyzed by MDH (but has been unable to do so in the reaction catalyzed by MR), so the structural factors important in the formation of this intermediate can be studied in the active site of MDH. He will also investigate the importance of matched pKa's for the donors and acceptors in anionic short, strong hydrogen bonds so that the requirements for the stabilization of enolic intermediates in enzyme active sites can be better understood.